Additionally, although the authors could separate Ile and Leu when standard solutions were analyzed, the amino acid pair was only partially resolved in Arabidopsis seeds extracts. In their same set of

experiments, the pair Thr and HSer always coeluted during chromatography regardless of the type of solution analyzed. To solve these problems, Gu et al. [10] used alternative MRM transitions for these pairs, but they were not as specific and sensitive for the respective amino acids as the transitions involving the most abundant fragment ions (for example refer to Figure 3 in ref [10]). It was demonstrated before by Petritis et al. [49] that selection of less abundant fragment ions caused a four- to six-fold Inhibitors,research,lifescience,medical loss of sensitivity for the LC-MS/MS analysis of native amino acids. It is worth noting that the lack of baseline separation [22] and irreproducible amino acid separation between standards and biological samples [36] have also been observed in the HPLC-ESI-MS/MS analysis of amino acids derivatized with FMOC, Inhibitors,research,lifescience,medical butanol, PrCl [22]

and TAHS [36]. As a result of the reproducible and satisfactory chromatographic separation of AQC amino acid derivatives obtained with the Inhibitors,research,lifescience,medical AccQ•Tag Ultra column in our studies, the development of our MRM-MS method was not complicated by overlapping elution of critical sets of amino acids, in contrast to previous observations with HPLC separation of native and derivatized amino acids. For example, it was not necessary to account for the crosstalking of 13C isotopes of Asn and Gln to the MRM channels of Asp and Glu, respectively, in order to accurately quantify these amino acids. Furthermore, Inhibitors,research,lifescience,medical there was no need to select additional Inhibitors,research,lifescience,medical fragment ions for the isomers/isobars, which may be less predominant and decrease the sensitivity of the transition channel used for MS detection of the corresponding amino acid. Additionally, reproducible chromatographic separation was obtained

in both amino acid standard solutions and sample extracts (see Figure 1 for example) and, therefore, quantitation of amino acids was straightforward. According to these results, the combination of AQC pre-column derivatization with the superior performance of UPLC selleck compound technologies allows reproducible separation of several critical amino acid pairs before MS/MS analysis, which is a necessity Vasopressin Receptor because of their similar nominal masses or identical fragmentation. This adds maximum selectivity and sensitivity to the amino acid analysis. 2.2.1. Method Evaluation The performance of the UPLC-ESI-MS/MS method for the analysis of AQC-derivatized amino acids was evaluated by measuring the repeatability, linearity, and sensitivity of the analysis. The repeatability of the method was determined by examination of the retention time and peak area ratios (i.e.